Ecotoxicology (2009) 18:522–536 DOI 10.1007/s10646-009-0310-9

Assessment of environmental contamination using feathers of Bubulcus ibis L., as a biomonitor of heavy metal pollution, Pakistan
Riffat Naseem Malik Æ Naila Zeb

Accepted: 6 April 2009 / Published online: 6 May 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Concentrations of metals such as Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Ni, Pb, and Zn were analyzed in the feathers of cattle egret (Bubulcus ibis) from three breeding colonies in the Punjab province, Pakistan. The mean concentrations of Ca, Cd, Fe, Pb and Mn were significantly different between the three study sites (River Chenab, River Ravi and Rawal Lake Reservoir). The mean concentrations of Ca, Cd, Fe and Mn were significantly greater at the River Chenab heronry and Cr, Co, Zn, and Pb concentrations at the River Ravi heronry. The feathers of cattle egrets collected from the Rawal Lake Reservoir heronry were least contaminated. Multivariate statistical methods viz., Factor Analysis based on Principal Component Analysis (FA/PCA); Hierarchical Cluster analyses (HACA), and Correlation Analyses identified relatively similar associations of metals and their sources of input. Metals such as Ca, Mg, and K were related with natural input from parent rock material whereas trace metals viz., Cu, Cd, Co, Pb, Ni, and Zn were associated mainly with anthropogenic processes. Metals such as Fe, Mn, and Li were either correlated with natural input or with anthropogenic activities. Concentration of heavy metals such as Cd, Pb, and Cr were well above the threshold level that can cause adverse effects in birds and pose menace to the cattle egrets population in Pakistan. The study suggested that the feathers of cattle egret could be used as a bio-monitor of the local heavy metals contamination. Keywords Heavy metals Á Multivariate analysis Á Biomonitoring Á Cattle egrets Á Feathers Á Pakistan
R. N. Malik (&) Á N. Zeb Environmental Biology Laboratory, Department of Plant Sciences, Quaid-i-Azam University, Islamabad 4600, Pakistan e-mail: r_n_malik2000@yahoo.co.uk

Introduction Heavy metals contamination is a great concern at global, regional and local level (Qadir et al. 2008) and influence the functional and structural integrity of an ecosystem (Qadir and Malik 2009). Heavy metals are ubiquitous, highly persistent, and non-biodegradable with long biological half-lives (Burger et al. 2007). Toxic concentrations of heavy metals affect the central nervous system and disrupt the functioning of internal organs (Lee et al. 2006). Among heavy metals, organic forms of mercury and arsenic do biomagnify in the food chain (Hoffman and Curnow 1979; De Luca-Abbott et al. 2001; Kojadinovic et al. 2007; Zolfaghari et al. 2007) and pose threats to the species at high trophic position with more bioaccumulation capacities. Heavy metals have been identified worldwide in diverse environmental compartments. Many studies have been carried out to investigate the level of their occurrence, accumulation and distribution (Burger and Gochfeld 1993, 2000c; Goutner et al. 2001; Kojadinovic et al. 2007; Kim and Koo 2007a; Qadir et al. 2008). These contaminants are added continuously to the pool of contaminants in the environment from anthropogenic as well as natural processes. Biological organisms and populations at different trophic levels are widely used as a biological indicator and/or bio-monitor to provide evidence of contaminant exposure and effects of one or more chemical pollutants (Covaci et al. 2002; Mateo and Guitart 2003; Dauwe et al. 2003, 2004; Martin-Diaz et al. 2005; Dural et al. 2006; Eeva et al. 2006; Kojadinovic et al. 2007). ‘‘Different species of birds such as herons and egrets have been used as a bio-indicator and/or bio-monitor of local environmental contamination because they are high on the food-chain, exposed to a wide range of chemicals, susceptible to bioaccumulation and are

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geographically widespread’’ (Burger 1994, 1995; Scheifler et al. 2006; Burger et al. 2007; Deng et al. 2007; Horai et al. 2007). Various studies have evaluated heavy metal accumulation in different body organs (Elliott and Scheuhammeri 1997; Mateo and Guitart 2003; Deng et al. 2007; Horai et al. 2007; Kojadinovic et al. 2007), blood (Scheifler et al. 2006), eggs (Fasola et al. 1998; Burger and Gochfeld 2004), eggshells (Ayas 2007), feathers (Fasola et al. 1998; Movalli 2000) and prey samples (Zhang et al. 2006) of different bird species. Studies using predatory migratory and non-migratory bird species provided valuable information on spatial and temporal trends of heavy metals (Burger and Gochfeld 2004). Higher concentration of heavy metals had been related with eggshell thinning, reproductive failure, and immuno-suppression, adverse effects on the developmental and malformations and lethality to embryos possibly leading to population decline (Spahn and Sherry 1999; Burger and Gochfeld 2000c). Heavy metals such as cadmium, mercury and selenium adversely affect the body mass/condition and health of birds by reducing their growth or body weight and have negative impact on survival and reproductive success (Dauwe et al. 2006). Morphometric parameters such as the body mass; tarsus length and wing length which correlate with concentration of Heavy metals had been used as indicators of survival and reproductive success of different species of birds. Chronic metal exposure can also produce detrimental effects on behavior, resistance to diseases, and other physiological mechanisms (Dauwe et al. 2005). Heavy metals are important factors in the formation of free radicals which causes oxidative stress, inhibit repair of DNA damage and form adducts in nucleotide bases. Higher oxidative stress may cause high mutation rates in birds (Bickham et al. 2000; Dahl et al. 2001; Eeva et al. 2006). Birds are generally exposed to heavy metals mainly by ingestion of food, drinking and by geophagy which is the practice of eating soil-like substances to obtain essential nutrients such as sulfur and phosphorus from the soil. Absorption rate varies among heavy metals depending upon intrinsic properties, species physiology, and bioavailability in particular media. Once metals are absorbed through the intestinal tract, these circulate through the body, get deposited in different body organs, excreted directly, or sequestered in feathers (Furness et al. 1986). Females also excrete some metals in their eggs and eggshells (Fasola et al. 1998). The use of feathers has been suggested as non-destructive means of assessing the contamination of heavy metals. These can be collected from live birds and particularly appropriate for rare and endangered species. Preservation of feathers does not require refrigeration and their metals contents profiles remain intact without any external contamination. The ratio of heavy metal concentrations in

feathers to other body tissues remains fairly consistent for most of the metals (Burger 1996). Feathers have been reported to reflect local contamination of an environment for young birds and non-migratory birds better than eggs, because birds sequester and excrete substantial amount of heavy metals in their feathers during the short period of plumage growth during molting. Heavy metals bind to the protein molecule in the feather during short period of feather growth when blood supply to the feather is intact (Veerle et al. 2004), thus concentration of heavy metals in feathers is a record of circulating blood level at the time of feather formation. Variation in heavy metals concentration in different feathers of an individual bird has been related to molting pattern, differences in pigmentation or external contamination. Fledging feather contaminant concentration has been used as a bio-indicator of heavy metal pollution of local environment (Fasola et al. 1998; Burger et al. 2007). Bird’s feathers have a potential in environmental studies to assess ecological health of the local ecosystem, and can be used as a non-destructive bio-monitoring tool. Recently, several studies have focused on the nondestructive techniques to monitor the concentrations of heavy metals and organic pollutants using hairs (Burger 1993; Covaci et al. 2002) and feathers (Pilastro et al. 1993; Nam et al. 2003; Dauwe et al. 2005; Jaspers et al. 2007; Van den Steen et al. 2007). For example, Dauwe et al. (2002) investigated concentration of two non-essential (Cd and Pb) and two essential (Cu and Zn) heavy metals in the outermost tail feathers of great (Parus major) and blue tits (Parus caeruleus) and Burger and Gochfeld (2000a) compared concentrations of Pb, Cd, Hg, As, Cr, Mn, Se and Ti in feathers of birds nesting on Midway Atoll in the northern Pacific Ocean. Heavy metal concentrations in the feather were also reported to be representative of long-term exposure to local contaminants (Kim and Koo 2007a). Attempts have also been made to assess and monitor heavy metals contamination in various environmental compartments in Pakistan (Movalli 2000; Boncompagni et al. 2003; Qadir et al. 2008). Movalli (2000) assessed heavy metal and other residues in feathers of Laggar falcon Falco biarmicus jugger from six districts of Pakistan and suggested that metal burden in adult and juvenile feathers reflected the concentration of contamination in particular districts. Boncompagni et al. (2003) monitored heavy metals contamination in three wetlands of Pakistan using eggs and feathers of colonial water birds such as little, intermediate and cattle egret. However, to our knowledge no such study has been conducted in our three study sites in close vicinity of freshwater aquatic habitats from Punjab province to assess the use of feathers of colonial water bird (cattle egret) for heavy metal contamination. This study was conducted to investigate heavy metal contamination in feathers of cattle egret (Bublus ibis)

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which feed mostly on grasshoppers, crickets, spiders, flies, frogs, and noctuid moths (Telfair 1994). It is medium sized bird, with a ‘hunched’ posture, even when it is standing erect and native to Africa and Asia, the most terrestrial heron, well-adapted to many diverse terrestrial and aquatic habitats belong to the family Ardeidae (Telfair 1994). This species has been found abundant in major rice growing tracts such as around Sukkur, Larkana and Hyderabad and cotton belts like Khanewal in Pakistan. The main objective of this research was to assess the heavy metals concentration in feathers, identify sources of main contaminants and to test null hypothesis that there were no differences in heavy metals concentration in the feathers of cattle egret from three sites. The results will provide baseline data for comparison with future metal concentrations in this species in the same and other localities and also help if the subject species is at risk due to heavy metal contamination of local environment.

Methodology Feathers from 4 to 6 day old chicks were collected from three different heronries (Rawal Lake reservoir, Chenab and Ravi Rivers) in the Punjab province of Pakistan (Fig. 1). The first heronry was located in the highly urbanized area of Islamabad City, the capital of Pakistan, in close vicinity to the Rawal Lake Reservoir at latitude 33° 410 2400 N and longitude 73°440 7300 E at an elevation of 520 m above sea level. The Rawal Lake Reservoir is of international significance and is protected within an isolated section of the Margalla Hills National Park (Malik and Husain 2006a, b, c; 2007; Bibi et al. 2008). It is a small water reservoir with some associated freshwater marshes, adjacent to a large area of protected woodland on the outskirts of Islamabad, in an area that forms the northeast part of the Potwar Plateau. It is the sole drinking water reservoir for the city of Rawalpindi and serves as an important habitat for wintering waterfowl (mostly Anas platyrhynchos). The lake is a partly ‘‘Arched Gravity’’ type reservoir with a discharge capacity of 2,300 m3/s and covers an area of 8.8 km2 with a maximum depth of 31 m. The lake has three primary inlets and one outlet. The Kurang stream enters on the northeastern side; Rumli and Quaid-i-Azam University streams enter on the northern side, while the spillway gates are located on the south-western side. The lake reservoir serves as an important resource for sports and commercial fishery. Fish yields in the lake have declined in recent years. It also is a popular area for outdoor recreation including boating and fishing. The climatic temperature at the Rawal Lake Reservoir varies from an average maximum in winter of 17°C to an average minimum of 3°C. In summer, the temperature varies from 24 to 34°C. The lake reservoir is facing

degradation of its water quality from point and non-point sources of heavy metal contamination which include surface runoff from agricultural and urban areas, waste from 360 poultry sheds in its catchments, recreational use of motorboats, car washes activities along the eastern margin of the lake and discharge from the feeding streams (such as Rumli, Shahdra, University stream), shoreline banks which fall into the reservoir, and recreational and human settlements in Bara Kahu, Malpur, Bani Gala and Noorpurshan villages and the Diplomatic Enclave, Islamabad. There is no treatment plant and raw sewage and municipal waste is discharged directly into the natural streams which feed the Rawal Lake Reservoir. The concentration of pollutants in the Rawal Lake Reservoir has increased in recent years. The other two heronries were located in the Khaniwal district. One was located in the close vicinity of Sardarpur village at latitude 30° 060 3400 N and longitude 71°810 4000 . This heronry was located about 1/2 km away from the River Chenab in cotton fields irrigated with water diverted from the river; whereas, the other heronry was located in the village Faqiranwala about one km from the River Ravi at latitude 30°620 6400 N and longitude 71°840 7800 E in cotton fields. The River Ravi is heavily polluted from industrial and municipal waste from the cities of Lahore, Kalashkaku and Qasur. Major anthropogenic sources of heavy metal contamination of the River Chenab include agricultural runoff, industrial and urban effluents from Sialkot, Gujrat, and Cheniot cities, and atmospheric deposition. From each study site, a total of ten nests were sampled and from each nest feathers from one chick were collected. Feathers from the Chenab and the Ravi were collected from mid June to early July; whereas, feathers from the Rawal Lake Reservoir were collected between early July and mid July 2007. A pinch of feathers from each side of the breast were plucked from each chick, placed in individual envelopes and labeled. All the feather samples were kept at room temperature for later laboratory analysis. Feathers were washed vigorously in de-ionized water alternated with acetone to remove the external contamination (Burger and Gochfeld 2000a, b), air dried overnight, and stored in metal free polypropylene vials. Each sample was weighed to 0.1 mg and digested in a Microwave Accelerated Reaction System (MARS, CEMÒ), in warm nitric acid mixed with the addition of 30% hydrogen peroxide, and subsequently diluted with de-ionized water. The metals such as Calcium (Ca), Cadmium (Cd), Cobalt (Co), Chromium (Cr), Cupper (Cu), Iron (Fe), Potassium (K), Lithium (Li), Magnesium (Mg), Manganese (Mn), Nickel(Ni), Lead (Pb), and Zinc (Zn) were analyzed using the Fast Sequential Atomic Absorption Spectrometer (Varian FS-AA-240). All samples were analyzed in batches with blanks and samples with known metal content, and a

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was used as distance Matrix and un-weighted pair group average (UPMGA) as linkage method. The mean concentration of metals in feathers was compared between the three study sites using the Analysis of Variance (ANOVA).

Results Metal concentration in feathers In general, high concentrations of the major metals such as Ca, Mg, and K, associated with natural origin, were measured in the feathers (Table 1). Among other metals, the highest means concentration was measured for Zn (142.5 ppm) followed by Fe (135.3 ppm), Pb (58.1 ppm), Mn (19.7 ppm), Ni (8.3 ppm), Co (6.6 ppm), Cr (6.5 ppm), Cu (3.9 ppm), Cd (2.7 ppm) and Li (0.8 ppm). The pattern of metal concentrations from the three study sites indicated that the Rawal Lake Reservoir and the Ravi River followed a similar pattern (Ca [ K [ Mg [ Zn [ Fe [ Pb [ Mn [ Ni [ Co [ Cr [ Cu [ Cd [ Li); while the metal pattern for the Chenab River site was slightly different (Table 1). Concentrations of Pb, Cd, Fe, Mn and Ca were significantly among sites (Table 1). The mean Pb concentration measured in feathers collected from the Chenab River heronry was significantly lower than mean concentrations measured in feathers collected from the Ravi River or Rawal Lake Reservoir heronries. Mean Pb concentrations in feathers collected from the Ravi River and Rawal Lake Reservoir heronries were not significantly different. The mean Mn concentration measured in feathers collected from the Chenab River site was significantly different from the mean the measured at the Ravi River and the Rawal Lake Reservoir sites; however, there were no differences the Rawal Lake Reservoir and the Ravi River sites. Similar site differences in Ca concentration were also observed (Fig 2). Concentrations of Cd measured in feathers collected at the Chenab River site were significantly different from concentrations measured in fathers from the Ravi and the Rawal Lake Reservoir. Identification of metals which contribute major variations in feathers Concentrations of most of the metals was highly correlated (Table 2). Three groups of association between metals were identified using HACA (Fig. 3). Group 1 consisted of K, Mg, and Ca. Group 2 consisted of Pb, Zn and Fe and Group 3 consisted of Cu, Cd, Cr, Co, and Ni. The metals Li and Mn were closely associated with Group 3 but were identified as outliers. Within Group 3, two sub-groups of heavy metals were identified, one sub-group consisted of Cu and Cd and other consisted of Cr, Co and Ni, respectively.

Fig. 1 Location of three study sites 1: Rawal Lake reservoir site; 2: River Ravi site and 3: River Chenab site

standard calibration curve. Standard Reference Material (SRM) was used for analytical precision, and quality assurance and control (QA/QC). Average values of three replicates were taken for each determination. The precision of analytical procedures was expressed as Relative Standard Deviation (RSD) which ranged from 5 to 10%, and was calculated from the standard deviation divided by the mean. The recovery rates for the selected metals were within 90 ± 10%. Chemicals, stock solutions, and reagents were obtained from Sigma/Fluka/Merck and were of trace metal grade. All glassware, was washed with distilled water, soaked in nitric acid (30%) overnight, rinsed in deionized water (Behropur B25), and air-dried. Statistical analysis The analytical results were compiled to form a multi-elemental database using Excel. Statistical analyses, including basic descriptive statistic, Correlation Analysis, Principal Component Analysis (PCA) based on Factor Analysis (FA), and Hierarchical Cluster Analysis (HACA) were performed using Statistics for windows software Kernel release ver. 5.5. Correlation analysis, HACA, and PCA/FA were performed on combined data from all three study areas. Correlations of metals were calculated using Pearson Correlation Matrix. In the PCA, the principal components (PCs) were calculated based on the correlation matrix. Varimax normalization was used as the rotation method in the analysis. The metals concentrations in the feathers samples varied greatly; therefore, the raw data was log transformed before the execution of clustering using HACA. The main objective was to identify metals with close correlation based on spatial similarities in different localities (Qadir et al. 2008). In HACA, Euclidean distance

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Table 1 Mean metal concentrations (ppm) measured in feathers of cattle egrets from three sites and n = 10 for each studied heronry Metals Cu Cr Mn Co Fe Ni Cd Zn Li K Pb Mg Ca CH 4.0 ± 1.4 6.6 ± 2.6 26.9 ± 12.2a 6.0 ± 2.1 181.8 ± 108.5 9.0 ± 3.5 3.1 ± 0.5a 133.8 ± 62.8 0.9 ± 0.3 2,400.0 ± 53.6 37.5 ± 10.7a 1,348.2 ± 266.3 23,962.0 ± 2,196.7a RV 3.7 ± 1.0 7.1 ± 2.2 15.3 ± 2.0b 7.6 ± 1.7 106.3 ± 30.0 8.1 ± 9.7 2.4 ± 0.6b 155.2 ± 102.3 0.7 ± 0.3 2,394.6 ± 50.4 76.5 ± 8.6b 1,292.8 ± 229.2 20,373.0 ± 3,966.7b RL 4.0 ± 1.3 5.38 ± 1.0 16.9 ± 7.1c 6.1 ± 2.2 117.7 ± 29.4 7.8 ± 11.8 2.7 ± 0.7 138.4 ± 49.2 0.8 ± 0.3 2,391.2 ± 41.7 60.2 ± 20.7c 1,272.9 ± 246.5 20,398.1 ± 4,019.8 Overall differences 0.784 0.533 0.008 0.165 0.039 0.700 0.050 0.798 0.271 0.919 0.000 0.782 0.045

Overall significant differences among sites are represented as italics and were tested by one way analysis of variance (ANOVA) CH Chenab River site, RV Ravi River site and RL Rawal Lake reservoir site a,b,c Differ significantly are marked using Mann–Whitney U test
Ca Mg K Pb Zn Fe Mn Li Ni Co Cr Cd Cu

Discussion The differences in concentrations of Pb, Mn, Fe, and Ca between the three study sites could be attributed to different local contamination/pollution sources and to accumulation of metals in the diet of cattle egret foraging in different localities. External heavy metal contamination of feathers can result either from deposition from the environmental contamination mainly from anthropogenic processes and/or excretion of the uropygial gland on the feathers during preening (Kim and Koo 2008). Exogenous contamination had been identified as one of the important source of heavy metals in feathers (Dmowski 1999; Veerle et al. 2004) and might be an important route of heavy metals in feathers (Dauwe et al. 2002). External contamination can result in a higher concentration of most of the heavy metals in feathers after their formation (Dauwe et al. 2003) and in particular to those segments of the feather that are exposed to the external conditions (i.e. atmospheric conditions and preening). De Luca-Abbott et al. (2001) indicated that diet had a significant effect on the concentration of contaminants accumulated in Ardeids. Burger et al. (2007) also reported that site differences in metal concentration could result from differences in local exposure, atmospheric deposition, or be related to foraging regimes of bird species. Previous studies reported Pb concentration in feathers of various birds’ species: Franklin’s gull (Burger 1996), pigeon guillemots (Burger et al. 2007), great tits and green finch (Deng et al. 2007), terek sandpiper, great knot, dunlin and Mongolian plover (Kim and Koo 2008). In the current study the mean Pb concentration in the feathers of cattle

Group 1 Group 2 Group 3

15

12.5

10

7.5

5

2.5

0

Euclidean Distance

Fig. 2 Hierarchical dendogram of metals obtained using UPMGA as linkage method and Euclidean distance matrix (the distances reflect the degree of association between different metals)

The results derived from the first five Principal Components/factors (PCs) of the PCA/FA indicated significant associations of different metals with each other (Table 3). The first five PCs with Eigen values[1.0 explained 77.32% of the total variance. The first PC extracted 27.5% of the variance, indicated correlation (P \ 0.05) with Mn, Cu, Fe, and Ca, and high loadings on of these metals along this axis indicated a tendency towards higher concentrations in feathers. The second PC which extracted 17.5% of the variance, was negatively correlated with Cu (r = -0.85), and Ni (r = -0.77). Third, fourth, and fifth PCs explained 14.7, 9.4 and 8.2% of total variance, respectively. The third axis indicated association with Co (r = 0.73) and Zn (r = 0.92), the fourth axis indicated a positive correlation with Cr (r = 0.80) and a negative correlation with K (r = -0.83), whereas, Pb was negatively associated (r = -0.90) as indicated by the fifth axis (Table 3).

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Use of feathers of Bubulcus ibis as bioindicator of heavy metal pollution, a case study from Pakistan Table 2 Correlation of contaminant levels in cattle egret (all sites combined) Cu Cu Cr Mn Co Fe Ni Cd Zn Li K Pb Mg Ca 1 -0.03 -0.23 -0.15 -0.31 0.44 -0.04 0.01 0.22 -0.08 -0.26 -0.17 0.10 1 -0.2 0.05 0.04 0.29 -0.18 -0.01 0.15 -0.47 -0.13 -0.2 0.11 1 0.23 0.88 -0.09 0.43 0.11 0.40 0.27 -0.43 0.57 0.55 1 0.23 -0.08 -0.09 0.53 0.14 -0.25 0.31 0.26 0.25 1 -0.02 0.35 0.03 0.46 0.16 -0.35 0.51 0.45 1 0.14 -0.22 0.36 -0.14 -0.22 0.01 0.36 1 0.04 0.06 0.42 -0.36 0.12 0.39 1 -0.04 -0.11 0.05 0.10 0.03 1 -0.16 -0.43 0.18 0.37 1 0.12 0.23 0.02 1 0.01 -0.30 1 0.58 Cr Mn Co Fe Ni Cd Zn Li K Pb Mg

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Ca

1

Given correlation are Pearson correlations. Correlation significant at P = 0.05 are highlighted and presented in italic

egret varied from 37.5 to 76.5 (lg/g) in three study sites (Table 4). Burger and Gochfeld (1993) reported mean Pb concentrations of 0.42 and 0.56 (lg/g) in the feathers of Pond heron and black-crowned night heron from China and Pb concentration of 9.1, 4.6, 4.4 and 1.5 (lg/g) in the black-crowned night heron, cattle egret, little egret and great egret from Hong Kong, respectively. Burger et al. (1992a) reported lower concentration of Pb that ranged from 0.23 to 9.7 ppm in cattle egrets. Mean Pb concentration was also very high compared to those reported in feathers of little egret (4.5 ppm) and black-crowned night heron (3.4 ppm) (Fasola et al. 1998), and in feathers of lagger falcon from six districts of Pakistan (Movalli 2000). Automobiles exhaust is probably the major source of airborne Pb in Pakistan (Movalli 2000; Qadir et al. 2008). Cattle egrets feed on insects along the road where Pb deposition is likely to be high (Burger and Gochfeld 1997). Lead concentration in feathers may be attributed to the continued use of leaded petrol in Pakistan which suggests its concentration in feathers mainly from external contamination. These findings strongly support those of Burger and Gochfeld (1993); Fasola et al. (1998) and Movalli (2000) that Pb concentrations in feathers are mainly attributed to the continued use of leaded gasoline. Information related to urban Pb emission is scarce in Pakistan. The results stresses further studies are required to determine if the vehicle emissions are affecting Pb concentrations in cattle egret population. Scheifler et al. (2006) indicated that Pb contained in insects or prey items of birds may become available to birds in local environments. Thus, Pb concentrations may partially be related to Pb concentrations in prey items of cattle egret; therefore, food transfer from soil invertebrates may also be an important route of Pb exposure for birds (Scheifler et al. 2006).

Approximately 87% of lead in the first primaries of blackbirds was of exogenous origin and 13% was attributed to internal lead from dietary uptake (Scheifler et al. 2006). The tail feathers may also reflect the body burden of lead and cadmium (Dauwe et al. 2002). High external contamination of tail feathers is probably related to lead-contaminated soil particles and the dust that is absorbed on plumage when the birds forage on the contaminated soils (Weyers et al. 1988; Kim and Koo 2007a). Dauwe et al. (2002) suggested that Pb concentrations in tail feathers could be due to either internal deposition via blood in growing feathers or to an external contamination due to excretion of the uropygial gland (i.e. preening). Results of the current a study indicated significant differences in Pb concentration between the three study sites. Several other studies have also reported significant differences in Pb concentration between the three study sites. Burger et al. (1992a) reported 41 times greater Pb concentration in feathers from Cairo than from Aswan, Egypt. Lead is a known as calcium-formations seeking element, readily accumulates in bones, hairs, feathers and nails and is not metabolically regulated which makes it potentially important for monitoring of anthropogenic pollution (Metcheva et al. 2006). It is a non-degradable metal (Scheifler et al. 2006) and its concentrations of 4,000 ppb (4 lg/g) in feathers were found to be associated with delayed parental and sibling recognition, impaired thermoregulation, locomotion, depth perception, feeding behavior, and lowered chick survival in gulls (Burger and Gochfeld 1993, 1995, 2000b; Burger et al. 1994). Lead (Pb2?) may interact with calcium (Ca2?) metabolism in birds (Hutton and Goodman, 1980). Pb causes behavioral deficits in animals due to its toxic effects on the nervous system, and may results in decreases in survival, growth rates, poorer fledging success,

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(a)

3.6 3.4 3.2

(b) 95
85 75

3.0

Cd (ppm)

Pb (ppm)
±1.96*Std. Err. ±1.00*Std. Err. Mean

2.8 2.6 2.4

65 55 45

2.2 2.0 1.8 RV CH RL 35 25 RV CH RL
±1.96*Std. Err. ±1.00*Std. Err. Mean

Sites

Sites

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34 30

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26000 25000 24000 23000

Mn (ppm)

26 22 18

Ca (ppm)
±1.96*Std. Err. ±1.00*Std. Err. Mean

22000 21000 20000 19000

14 10 RV CH RL

18000 17000 RV CH RL

±1.96*Std. Err. ±1.00*Std. Err. Mean

Sites

Sites

(e)

300

260

220

Fe (ppm)

180

140

100
±1.96*Std. Err. ±1.00*Std. Err.

60 RV RL CH

Mean

Sites

Fig. 3 Box and Whisker plots of metals significantly different in three studied heronries, CH = Chenab, RV = Ravi and RL = Rawal Lake a) Cd, b) Pb, c) Mn, d) Ca, and e) Fe

learning, and metabolism (Dauwe et al. 2005; 2006). Higher incidence of mortality among the lead-exposed nestlings was found (Spahn and Sherry 1999). Pb concentration reported in the current study is well above the threshold level known to cause adverse effects in the birds (Burger and Gochfeld 2000c; Burger et al. 1994). The mean Cd concentration measured in cattle egret feathers collected from the Chenab River heronry was significantly greater than the mean concentrations measured in cattle egret feathers collected from the Ravi River or Rawal Lake Reservoir heronries (Table 1). Mean Cd concentrations in feathers collected from the Ravi River and Rawal Lake Reservoir heronries were not significantly different. Cadmium concentrations measured in feathers in the current study were greater than concentrations reported in feathers in most other studies (Table 4). Greater

concentrations of Cd have been reported in feathers collected from birds in Korea, North Pacific Ocean, Belgium (Honda et al. 1986; Burger and Gochfeld 2000a; Dauwe et al. 2002; Table 4). Cadmium concentrations (2.4–3.1 lg/g) in feathers reported in the current study was somewhat greater to those reported for little egret (0.6 ppm) and black-crowned night heron (0.6 ppm) in the northern Italy (Fasola et al. 1998), 0.18 and 0.22 (lg/g) for Pond heron, and black-crowned night heron from China, and 0.14, 0.43, 0.05 and 0.07 (lg/g) for black-crowned night heron, cattle egret, little egret, and great egret from Hong Kong (Burger and Gochfeld 1993). Similarly, Cd concentrations measured in the current study were much greater compared to those reported in the tail feathers of 15-day-old great tits nestling (0.2 lg/g) along a pollution gradient in Belgium (Dauwe et al. 2004).

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Use of feathers of Bubulcus ibis as bioindicator of heavy metal pollution, a case study from Pakistan Table 3 Values of five extracted factor loadings for the studied metals; values of marked loadings [.70 are significant at p = 0.05 in each factor Metals Cu Cr Mn Co Fe Ni Cd Zn Li K Pb Mg Ca Eigen value Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 -0.22 0.01 0.73 0.40 0.75 0.24 0.23 -0.03 0.46 0.15 -0.08 0.83 0.78 3.58 -0.85 -0.04 0.26 0.07 0.33 -0.77 -0.07 0.04 -0.29 0.10 0.18 0.05 -0.33 2.28 17.54 0.09 -0.10 0.12 0.73 0.02 -0.26 -0.01 0.92 0.01 -0.22 0.10 0.07 0.05 1.91 14.69 -0.06 0.80 -0.19 0.21 0.03 0.23 -0.49 -0.03 0.34 -0.83 -0.13 -0.23 0.01 1.22 9.38 0.14 0.06 0.50 -0.29 0.45 0.05 0.56 0.07 0.41 0.02 -0.90 -0.16 0.18 1.07 8.20

529

Total variance (%) 27.5

Cadmium concentrations measured in the current study may partly be attributed to dietary Cd concentrations. Kim and Koo (2007b) reported that cadmium contents in feathers of wild birds increased in relation to cadmium concentrations in the diet. Greater concentrations of Cd in herons and egrets have also been associated to contamination in their feeding grounds (Kim and Koo 2007a). Higher Cd concentration in feathers and sediments indicates its wide distribution in the environment which consequently deposited into feathers. Cadmium does tend to bio-accumulate in food chain (Burger and Gochfeld 2004). Toxicological effects of Cd in birds have been reviewed by Furness (1996). Though, it is toxic above certain concentrations, Cd is not an essential element for animals and may induces deficiencies of essential elements through competition at active sites in biologically important molecules. At higher concentrations it may causes kidney damage, altered behavior, suppression of egg production, egg shell thinning, and testicular damage (Furness 1996). At the population level, reduced growth rates of bones and fledgling success were correlated with exposure to elevated Cd concentration in feathers (Spahn and Sherry 1999). Cadmium causes sub-lethal and behavioral effects at lower concentrations than mercury or lead (Burger and Gochfeld 2000c). Cadmium concentration above 100 ppm in kidneys has been suggested as a threshold concentration, above which Cd poisoning can be expected (Furness 1996). Burger and Gochfeld (2000c) considered Cd concentration of 2,000 ppb (2 lg/g) as a threshold concentration in feathers that may have adverse effect in kidneys. The mean Cd concentration measured in egret feathers in the current study was above the threshold

concentration of 2 lg/g which may cause potential adverse effects in birds. The results indicated similar site differences of Mn concentration (an essential micronutrient, serves as an important cofactor in general body mechanisms) as measured for Cd. Feathers from the River Chenab site were significantly different in Mn concentration to those from the Ravi River and the Rawal Lake Reservoir sites, however, there were no significant differences between the River Ravi and the Rawal Lake Reservoir sites. Mean Mn concentration (15.3– 26.9 lg/g) in feathers of cattle egret from three sites were within the range reported for little egrets nesting (17.5 lg/g) from Hong Kong, and for pond heron (23.5 lg/g) from China (Burger and Gochfeld 1993). However, these concentrations were greater to those reported for little egret (2.7 ppm), intermediate egret (2.2 ppm) from the Haliji Lake wetland, Pakistan (Boncompagni et al. 2003), common eider (2.6 and 1.2 lg/g) and tufted puffin (0.7 and 0.5 lg/g) from the Aleutian Chain of Alaska (Burger and Gochfeld 2008) and lower to those reported for pigeon guillemots (0.9–1.75 lg/g) collected from the Alaska (Burger et al. 2007). Similarly, Nam et al. (2005) and Burger and Gochfeld (2000a) also exhibited lower mean Mn concentrations for feathers of adult great cormorant (8.8 lg/g) from Japan, adult red-footed boobies (1.5 lg/g), and great frigate-birds (0.6 lg/g) from Midway Atoll, Northern Pacific Ocean. Mean Mn concentrations measured in the current study was lower to those reported for cattle egret (36.6 lg/g) from Hong Kong. Presence of Mn concentration in the feathers could be coupled to the concentrations in the contaminated ingesta and to some extent to air (Hui 2002), however, inhalation also provides small Mn exposure. Burning of diesel fuel is one of the sources of atmospheric Mn which is used as an anti-knocking agent in leaded gasoline (Qadir et al. 2008). Manganese is regulated in birds primarily by excretion in the feces, however; hens also excrete Mn in eggs. Teratogenic effects (such as micromelia, twisted limbs, hemorrhage, and neck defects), behavior impairments, altered growth rates and reduction of hemoglobin formation have been linked to sub-lethal Mn exposure in animals and avian embryos (Burger and Gochfeld 1995). Manganese tends to accumulates in bone, liver, pancreas and kidney of avian species. However, the potential effects of Mn on egrets are unknown indicating that a detailed study is required, as it is used in gasoline. Table 4 indicated that mean concentration of Cr measured in the feathers in the current study was lower than to those concentrations reported from New York, Delaware, Puerto Rico, and Egypt, china, and Hong Kong in feathers of various bird species (Burger et al. 1992b; Burger and Gochfeld 1993, 1995). However, mean Cr concentration measured in the current study in cattle egret is higher than the little egret from Taunsa, Karachi and Haliji wetland,

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530 Table 4 Comparison of mean concentration of metals (lg/g) in feathers reported in the present study with literature
Authors Present study Study area Chenab river Pakistan Ravi river Pakistan Rawal Lake reservoir Pakistan Burger and Gochfeld (2008)a Amchitka Species Cattle egret Cattle egret Cattle egret Common eider Cd 3.1 2.4 2.7 0.08 Cu 4.0 3.7 4.0 Pb Zn Cr Ni Co Mn Fe Li K

R. N. Malik, N. Zeb

Mg

Ca

37.5 133.8 6.6 76.5 155.2 7.12 60.2 138.4 5.83 0.83 0.10

9.0 6.0 26.9 8.1 7.6 15.3 7.8 6.1 16.9 2.56

181.8 0.9 2,400

1,348.2 23,962

106.3 0.7 2,394.6 1,292.8 20,373 117.7 0.8 2,391.2 1,272.9 20,398.1

Kiska Amchitka Kiska Kim and Koo (2008) Yeongjong Island, Korea

Common eider Tufted puffin Tufted puffin Kentish plover

0.08 0.10 0.05 0.9 9.4

1.15 0.93 1.68 9.8 88.4

0.24 2.04 1.52

1.18 0.70 0.53 4.4 2.6 8.5 6.2 4.6 2.5 0.0 7.1 2.5 0.0 7.1 10 12.2 150 86

Mongolian plover 0.4 Dunlin Great knot Terek sandpiper Deng et al. (2007) Badachu Park, China Kim and Koo (2007b) Burger et al. (2007)a Korea Great Tit Greenfinch Black-crowned night heron Grey heron Prince William Sound Amchitka Kiska Scheifler et al. (2006) Urban area 0.1 0.0 174 172 1.1

10.4 20.7 67.9 8.2 4.1 2.8 8.1 14.8 103.0 20.8 71.5 87.1 276.0 2.2 276.6 1.5 29.6 36.5 1.33 0.80 2.69 3.4 7.4

10.4 3.3

14.2 0.5 9.1 0.4 1.02 0.90 1.72 3.2

Pigeon guillemots 0.099 Pigeon guillemots 0.03 Pigeon guillemots 0.028 Blackbirds Little egret Little egret Little egret Adult great cormorants Great tits Little egret Intermediate egret 0.0 0.2 6.7 3.0

1.75 0.90 1.24

Zhang et al. (2006) Poyang China Tai China Pearl Delta China Nam et al. (2005) Dauwe et al. (2004) Boncompagni et al. (2003) Japan Belgium Haleji lake Pakistan

206.0 0.6 193.0 0.8 18.7 1.7 2.6 0.6 0.1 8.8 0.3 2.7 2.2 161.0 0.3 54.6 154.0 1.2 214.0 0.2 199.7 0.9 0.4 1.8 11.6 8.0 88 69 250 271 0.98 240.0 244.0 2.5 1.46 0.5 0.7

Karachi Pakistan Taunsa Pakistan Dauwe et al. (2002) Burger and Gochfeld (2000a) Dauwe et al. (2000) Movalli (2000) Belgium

Little egret Little egret Cattle egret Great tit Blue tit

Northern Pacific Ocean

Red-footed booby 51.3

Great frigate bird Polluted site Belgium Blue tit Great tit Bahawalnager Pakistan Bahawalpur Pakistan Chachero Pakistan Jacobabad Pakistan Karachi Pakistan Mithi Pakistan Eens et al. (1999) Belgium Laggar falcon Laggar falcon Laggar falcon Laggar falcon Laggar falcon Laggar falcon Great tit Blue tit

204 0.1 0.1 0.1 0.1 0.1 0.1 0.0 0.1 2.6 3.0 5.1 5.8

1.50 3.7 0.5 1.1 2.8 1.1 1.3 0.0 0.9 119.0 127.0

1.1

0.59

104.0 1.7 113.0 1.7 0.0 0.0 0.0 0.0

1.1 1.1 0.0 0.0 0.6 0.7

104.0 2.3 111.3 2.4

13.8 16.3 172.7 24.0 64.0 252.6

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Use of feathers of Bubulcus ibis as bioindicator of heavy metal pollution, a case study from Pakistan Table 4 continued
Authors Spahn and Sherry (1999) Fasola et al. (1998) Study area South Louisiana (Bueche contaminated site) Northern Italy Species Little blue heron chicks Little egret Black-crowned night heron Burger and Gochfeld (1997)a Sulawesi Cattle egret Cd 0.25 0.6 0.6 0.36 Cu Pb 1.01 4.5 3.4 3.58 0.70 5.94 Zn Cr Ni Co Mn Fe Li K Mg Ca

531

Bali Bali Bali Burger and Gochfeld (1993) Szechuan, China

Cattle egret Little egret Pond heron

0.24 0.75 0.2

2.72 3.53 3.67 4.2

0.60 0.58 0.64 49.1

1.93 2.57 1.67 23.8

Intermediate egret 1.94

Black crowned night heron Honk Kong Black-crowned night heron Cattle egret Little egret Great egret Burger et al. (1993)a Honda et al. (1986) a 0.2 0.1 0.4 0.0 0.1 0.21 0.20 4.7

5.6 9.1 4.6 4.4 1.5 2.23 3.64 0.3 63.3

20.8 17.1 16.1 18.9 8.5 15.80 10.00 0.2

42.4 45.1 36.6 17.5 4.2 11.99 4.98 2.0 31.4

Costa Rica Florida Korea

Wood stork Wood stork

Egretta alba 13.0 modesta chicks

Conversion from ng/g, ppb to lg/g

intermediate egret from Haliji, and cattle egret from Tunassa barrage wetland, Pakistan (Boncompagni et al. 2003), red-footed booby and great frigate bird from Midway Atoll, Northern Pacific Ocean (Burger and Gochfeld 2000a), Laggar falcon from different districts of Pakistan (Movalli 2000) and Franklin gulls in the United States (Burger 1996). The results indicated non-significant site differences of Cr concentration. ´ ´ According to Kertesz and Fancsi (2003) Cr produces adverse effects on the embryonic development, hatching and viability of the mallard. According to Burger (1993) and Burger and Gochfeld (2000c) Cr concentration of 2,800 ppb (2.8 lg/g) in birds feathers might be associated with adverse effects. Using this concentration as a threshold, our result indicated that the population of cattle egret is at risk as Cr concentration was high. The mean Zn concentration in the current study (133.8 lg/g from the river Chenab site, 138.4 lg/g from the Rawal Lake Reservoir and 155.2 lg/g from the River Ravi site) was lower than to those found in the study of Boncompagni et al. (2003) in feathers of little egret, and intermediate egrets, and greater to those for feathers of cattle egret from Taunsa wetland of Pakistan (Table 4). Similarly, the mean Zn concentration was also lower to

those for feathers of little egret from the Poyang and Tai wetlands of China (Zhang et al. 2006), great and blue tits from Belgium (Eens et al. 1999; Dauwe et al. 2002), and adult great cormorants from Japan (Nam et al. 2005). Doi and Fukuyama (1983) found Zn concentrations ranged 50–250 ppm for the feathers of wild and zoo-kept birds in the Hokkaido, Japan. The mean Zn concentration measured in feathers of cattle egret were greater than the mean concentrations measured in feathers of laggar falcons collected from six districts in Pakistan (Movalli 2000), Egretta alba modesta Chicks from Korean (Honda et al. 1986), sparrow hawks, barnowls, and little owls from Belgium (Dauwe et al. 2003), and great tits nestling along pollution gradient in Belgium (Dauwe et al. 2004). However, the mean Zn concentration in the feathers of cattle egret did not differ significantly between the sites. Mean differences in Zn concentration were small between the three study sites. Dauwe et al. (2002) also revealed that zinc contamination between the polluted and non-polluted sites is relatively low. Zinc is an essential heavy metal required for normal feather formation and is essential for proper body functioning and provides protection against the renal toxicity of Cd. However, the results of the current study found no correlation between these two elements. The results

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532

R. N. Malik, N. Zeb

indicated that Zn concentration in cattle egrets was still lower than the concentration leading to pathological kidney damage (200 mg/dry g; Hutton and Goodman 1980). Similarly, non-significant site differences of Co, Cu and Ni concentrations were recorded. The mean concentration of Co which is an activator of many enzymes varied from 3.20 to 9.40 (lg/g) in three sites and these concentrations were greater than to the mean concentrations in Laggar falcon from Karachi, Mithi, and Bahawalpur districts, Pakistan (Table 4). Similarly, Nam et al. (2005) also found lower mean Co concentration in feathers of adult great cormorants from Japan. Copper is an essential for normal growth, metabolism for living cells and structure and function of many proteins vital for cell functioning (Pappas et al. 2006). The mean Cu concentration measured in feathers of cattle egret in the three study sites was within the range reported for other bird species. However, Cu concentration measured in the current study (Table 4) was lower than to those for feathers of Kentish and Mongolian plover, great knot and dunlin from Yeongjong Island, Korea (Kim and Koo 2008), green finch from China (Deng et al. 2007), grey heron and black crowned night heron from Korea (Kim and Koo 2007b), adult great cormorant from Japan (Nam et al. 2005), tail feathers of blue and great tits nestling from polluted sites in Belgium (Dauwe et al. 2000) and greater than to those in the feathers of great tits from the Badachu park China (Deng et al. 2007). Nickel is related to the pigmentation of feathers in birds and excreted via the feathers by moulting (Honda et al. 1986). The mean Ni concentration varied between 7.8 and 9.0 (lg/g) which was very high to those of the mean Ni concentration in the feathers of great tits from four sites in Belgium and great tits at Badachu Park in the Western Mountains, Beijing, China (Deng et al. 2007). The above findings suggested that the cattle egrets of three colonies may be exposed to relatively high Ni concentration or potentially toxic concentrations of Ni contamination. Among naturally occurring metals (such as Ca, Mg, Fe and K) mean concentration of Ca was measured greater followed by the concentration of K, Mg, and Fe. The mean Ca concentrations measured in feathers of cattle egret collected from the Chenab River heronry was greater than the mean concentration measured in the feathers from the Rawal Lake Reservoir heronry. Similar trend was observed for concentration of Mg, however, the mean concentrations of Fe and K measured in feathers collected from the Ravi River heronry were lowest (Table 4). The mean concentrations of Fe and Mg measured in the current study in cattle egret feathers was greater than to the mean concentrations measured for feathers of Nestling Ospreys in the Chesapeake Bays (Rattner et al. 2008).

A number of studies used different multivariate techniques such as HACA, PCA/FA and correlation analysis to evaluate spatial variations, source identification of pollutants, interpretation of complex environmental data matrices, and to assess ecological status of studied systems. These methods can be used as valuable tool for effective management of ecosystems to solve problems related to environmental contamination (Shrestha and Kazama 2007; Qadir et al. 2008). The results of the present study also demonstrated the usefulness of multivariate techniques such HACA, PCA/FA and Correlation analysis in assessment of contamination of feathers and identification of possible sources of different metals. Pearson product– moment correlation analysis was used to test whether the mean concentration in the feathers was correlated with the molting sequence within three bird species (Dauwe et al. 2003). Kim and Koo (2008) used Pearson correlation analysis of heavy metal concentrations between livers and feathers of five shorebird species from Yeongjong Island, Korea in the East Asian–Australian migration flyways. Pain et al. (1999) used correlation analysis between different contaminants such as Hg and pesticides and egg shell thickness to ascribe causality of effect. The results demonstrated the useful of multivariate techniques in assessing the source of metal contamination. In the current study, the results of HACA agreed well with that of the PCA/FA. Both techniques illustrated similar sources of metal input in feathers. Based on PCA/FA and HACA, three main sources of metals can be recognized. The first group of elements consisted of Ca, Mg, and K separated from the other metals in HACA indicated their common origin. The results of PCA/FA also highlighted that Ca and Mg along with Mn and Fe had strong correlation with PC1 which explained a total variance of 27.5% and K with PC4 with high significant correlation (r = -0.83) may explain natural geochemical association of these elements with parent rock material. According to Shrestha and Kazama (2007) varifactors from factor analysis generally indicates that parameters that are mainly related to different point and non-point sources of pollutants. Correlation analysis strongly supported theses results as significantly positive correlation was found between Mn, Fe and Li which may reflect their input either from parent rock material or related with anthropogenic activities. Mn and Fe indicated highly significant association with PC1 indicating their input can be related with parent rock material. However, Fe was clustered with Pb, Zn in HACA indicating that other possible source of these metals could be related with anthropogenic activities. Fe, Mn and Li, alkaline metal elements, accumulated higher in terrestrial birds than in aquatic ones, especially ocean species (Horai et al. 2007) reflected strong positive correlation (r for Mn

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Use of feathers of Bubulcus ibis as bioindicator of heavy metal pollution, a case study from Pakistan

533

and Fe = 0.88, Fe and Li = 0.46 and Li and Mn = 0.40) with each other indicating their common source of origin. Horai et al. (2007) also indicated that Mn is emitted into the environment through the combustion of fossil fuels, which is one of the main anthropogenic activities. The potential use of Mn in gasoline makes it critical to have baseline information on concentrations in biota (Burger and Gochfeld 2004). Horai et al. (2007) also suggested that the distributions of Mn in body of birds may also be affected by diet. Cu, Cr, Co, Cd, Ni, Pb, and Zn were derived from anthropogenic sources mainly related with industrial sources, domestic combined with traffic activities. Similarly, the result of PCA/FA, HACA and Correlation Analysis also indicated the main sources of input of these metals are mainly from human related activities. A number of studies have reported the input of heavy metals clustered in group 2 and 3 from similar sources and related their sources to anthropogenic input (Qadir et al. 2008). Qadir and Malik (2009) and Qadir et al. (2008) also revealed that contamination of Cu, Pb and Zn are mainly related with anthropogenic input. These findings are consistent with the observations from other studies that have reported higher accumulation of some heavy metals such as Pb, Zn and Cd in urban soils (Fakayode and Olu-Owolabi 2003). Moreover, Dauwe et al. (2002) also reported significant correlation between Pb and Cd concentrations in the great tit feathers and suggested that external contamination onto the feathers surface of great tit may be an important route of heavy metals and found no significant correlations between Cu and Zn concentrations. Similarly, Dauwe et al. (2003) indicated that concentration of Al, Cd, Co, Fe, Mn, Ni, Pb and Zn measured in feathers of birds of prey might partially be from external deposition related with various anthropogenic activities. Veerle et al. (2004) also found similar results that the concentration of Cd, Co, Cu, Fe, Mn, and Pb might be partially due to exogenous contamination. A number of studies have used heavy metal concentrations in various types of feathers for bio-monitoring purposes (Eens et al. 1999; Ayas 2007; Dauwe et al. 2003; 2004; 2005). The results of the present study strongly support the use feathers of cattle egret as ecological indicator of local contamination and conclude that heavy metals in feather of cattle egrets can be used as an indicator of exposure and to track changes in contaminant concentrations from local environment. These findings further underline the potential usefulness of bird’s feathers as a routine monitoring tool over time (e.g., seasonally) and space. Furthermore, the results of the current study stress a dire need to establish a non-destructive monitoring method for contaminants in birds, especially for endangered species.

Conclusions Different multivariate statistical techniques used in this study demonstrated their utility in assessing metal concentration; identifying major contaminants and their possible sources in feathers of cattle egret from three different localities in Punjab province of Pakistan. The results of PCA/FA and HACA indicated significant metals relationships which indicated their common sources of input in the study area. The association of metals such as Cu, Cd, Cr, Co, Ni, Pb, and Zn is mainly related with anthropogenic activities. Association of K, Ca, and Mg are attributed to parent rock material whereas Fe, Mn, Li may reflect the inputs from some anthropogenic activities and/or natural geochemical system. The results of the present study reinforce the idea that high concentrations of heavy metals reflected in the feathers of cattle egrets due to local environmental exposure. Cattle egret is geographically widespread species and high on the food chain and may facilitate comparisons among different localities and its feathers can be useful tools for environmental bio-monitoring studies and should be useful for long-term evaluation of potential environmental hazards. Since, feathers are easy to collect and their collection method does not harm the chicks, it can be a practical method to use feather of cattle egret as the effective bio-indicators of heavy metals.
Acknowledgments The authors are highly thankful to Mr. Ali Mustajab and Ms. Sidra Raouf, research students in the Environmental Laboratory, Quaid-i-Azam University, Islamabad for their assistance in the field work and Mr. Tanweer for his help in the metal analysis. The authors are grateful to Pakistan Wetlands Programme (PWP) for providing transport during River Chenab sampling during 2007 when two heronries on River Chenab and Ravi were identified. Research work was done when Ms. Naila Zeb was an M.Phil student under the supervision of Rifffat N. Malik in Environmental laboratory, QAU. The authors also extend their thanks to Mr. M. Nadeem and Z. A. Malik, M.Phil students in Environmental Biology Laboratory, for checking the references of this manuscript. We also thank Dr. Rahat Bokhari and his friend for English correction and extend thanks to two anonymous reviewers for their constructive comments to improve the quality of this manuscript.

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